Complete Genome Sequence of a Carbapenem-Resistant Escherichia coli Isolate with bla NDM-5 from a Dog in the United States.

The carbapenem resistance gene bla NDM-5 was identified in an Escherichia coli strain isolated from a dog. We report here the complete genome sequence of this E. coli strain; the bla NDM-5 gene was present on a large IncFII multidrug-resistant plasmid. This is the first bla NDM-5-carrying E. coli strain from an animal in the United States.

ANNOUNCEMENT

Carbapenems are a class of beta-lactam antibiotics that are active against many aerobic and anaerobic Gram-positive and Gram-negative organisms. They are critically important antibiotics used to treat serious bacterial infections (1). Carbapenem resistance is rare and typically results from expression of a carbapenemase that hydrolyzes penicillins, cephalosporins, monobactams, and carbapenems. Known genes include blaKPC, blaOXA, blaVIM, and blaNDM, among others, and have been found in various Gram-negative organisms (2–4).

In July 2018, Escherichia coli strain 24213-18 was isolated from an endotracheal wash specimen obtained from a dog in a veterinary hospital in Philadelphia, Pennsylvania. The isolate was phenotypically resistant to imipenem (MIC, 4 μg/ml), and short-read sequencing identified the carbapenem resistance gene blaNDM-5. As a result, the isolate was sequenced by long-read technology.

The isolate was grown on blood agar at 35°C for 24 h. Genomic DNA was extracted with the DNeasy blood and tissue kit (Qiagen, Germantown, MD), quantified on the Qubit fluorometer with the Qubit double-stranded DNA (dsDNA) broad-range (BR) assay kit, and fragmented to 10 kb by using a G-tube. The quality and size of DNA were measured by FEMTO Pulse. The DNA library, ligated with a symmetrically paired barcode, was prepared following the 10-kb template preparation protocol with SMRTbell template prep kit v 1.0. Whole-genome sequencing (WGS) was performed on a Pacific Biosciences Sequel sequencer with Sequel sequencing kit v 3.0. Sequences were collected after 120 min of preextension for 600 min of movie length.

There were 65,426 polymerase reads with a 41,595-bp mean read length and 620,057 subreads with an N50 value of 6,330 bp. The reads were filtered with high-quality read filtering at the primary analysis step, to ensure that only single reads longer than 50 bp per zero-mode waveguide were used, and were de novo assembled by the PacBio Hierarchical Genome Assembly Process (HGAP) v 4.0 program. Each assembled contig had a mean confidence quality value above 90 (one error in 1 Gb). The contigs were further circularized with Circlator (5) with the default parameters. The sequences were annotated on the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v 4.8 (6).

The total genome size was 5,135,313 bp with a GC content of 50.8%. Assembly resulted in a single circular chromosome of 4,857,938 bp, with 93× coverage, and two plasmids. WGS-based multilocus sequence typing (MLST) (7) determined that this E. coli strain was sequence type 167 (ST167), the same sequence type as that of dog isolates with blaNDM-5 from Finland (8). There was a circular IncFII plasmid of 139,547 bp with 93× coverage that contained the blaNDM-5 gene and the additional resistance genes tet(A), aac(6′)-Ib-cr, aadA5, aadA2, blaOXA-1, blaCTX-M-15, catB3, dfrA7, dfrA12, sul1 (two copies), and mph(A). These genes confer resistance to various drug classes, and confirmed phenotypic testing on a Vitek 2 system (bioMérieux) using the GN65 panel showed resistance to tetracycline, aminoglycosides, beta-lactams, chloramphenicol, fluoroquinolones, and trimethoprim-sulfamethoxazole. This plasmid is highly related to plasmid p28078-NDM found in E. coli from China (GenBank accession number MF156713). The additional contig was not circularized, and it was an IncI plasmid of 137,828 bp with 91× coverage and resistance genes blaTEM-1D, floR, ant(3″)-Ia, and aac(3)-IId.

Data availability.

The assembled genome sequence for this isolate was submitted to GenBank under accession numbers CP041392, CP041393, and CP041394. Raw sequence reads were deposited into the SRA under accession number SRR9668343.